Multi-chambered pump-valve device

ABSTRACT

A multi-chambered pump-valve device for performing chemical processes, detections or analyses is described herein. The device includes a plurality of chambers having variable volumes in fluid communication with one another via one or more passageways. Liquid may be directed through the device by merely changing the volumes of two or more chambers. Despite the simplicity of the mode for transferring liquids in the device, complex chemical processing sequences may be performed using the device. A plurality of devices may be incorporated into a larger apparatus so that a plurality of chemical processing operations may be performed substantially simultaneously in parallel.

BACKGROUND OF THE INVENTION

Modern chemical (including biochemical) practice includes numeroustechniques for treating a liquid sample: mixing, reacting, filtering,dialyzing, synthesizing, fractionating, detecting, catalyzing (includingenzymatically catalyzing) reactions, performing various separations, andthe like.

For example, it may be desirable to remove solvent from a liquid samplein order to concentrate one or more solutes so that they may beanalyzed, detected, further treated, etc. It also may be desirable toremove solutes, including macromolecular solutes or low molecular weightions, each of which may interfere with analysis, detection or furthertreatment of one or more solutes of interest. This may be particularlyso in biochemical practice, where complex mixtures of biologicalmolecules can be obtained from living organisms, especially when thepresence of a particular chemical species may interfere with thedetection, analysis or further treatment of other solutes in themixture.

Standard chromatographic techniques may be useful for performing manytypes of chemical separations. In addition, modified chromatographicmethods in which a sorptive or reactive medium is cast in-place in astructure such as a pipette tip may be used to perform chemicalseparations on microliter-volume samples. In contrast to standardchromatographic methods, such modified, microliter-volumechromatographic methods can include a device that permits a sample to besubjected to multiple passes through the sorptive or reactive medium,thereby allowing multiple opportunities for the chemical separation toprogress so long as the sample does not saturate the sorptive orreactive capacity of the medium. However, such devices may not beconvenient for chemical separations in samples of larger volumes, e.g.,milliliter or multiple-milliliter volumes.

Certain chromatography systems include a plurality of pumps to supplysolvent to a plurality of chromatography columns. Each pump includes achamber with a movable piston, an inflow valve and an outflow valve.Withdrawing the pistons from the chambers generates a vacuum in eachchamber that draws solvent through the inflow valve and into thechamber. When the desired delivery volume of solvent has been drawn intoeach chamber, the pistons may be pushed back into the chambers, therebyforcing the solvent through the outflow valve and into thechromatography column. The pistons may be driven in concert using apneumatic or hydraulic system.

Some devices designed for continuous chromatography have been adapted tobe useful for the purpose of treating a liquid sample by depleting oneor more undesirable chemical species. In such adaptations, thechromatography column is replaced by an element containing a substanceselected to perform the desired depletion treatment, such as afunctionalized solid support. The samples requiring treatment may beintroduced into the buffer stream, thereby allowing the chemical speciesthat are to be depleted from the sample to interact with the depletionelement. Such devices may have at least two reservoirs, one for carryingthe liquid sample, and a second for generating the column. In suchdevices, a second buffer can be used to elute the separated species fromthe depletion element so that the depletion element may be used to treatone or more subsequent samples.

Alternatively, certain batch processes are known for depleting anundesirable chemical species from a liquid sample. In such processes, asmall chamber may include an article that contains a depletion substanceselected to perform the desired depletion, i.e., removal of at least aportion of an undesirable chemical species from the liquid sample. Asample may be flushed through the chamber using, for example, low speedcentrifugation or syringe plunger pressure, thereby allowing thedepletion substance to remove the undesirable chemical species from thesample. For complete depletion, it may be necessary to pass the samplethrough the depletion substance more than once, thus requiring theoperator to collect the partially depleted sample and recycle it throughthe process.

Certain other devices are designed for extraction of nucleic acids froma sample without pipetting. The sample and a lysis buffer arepredispensed in vessels such as syringes that are interconnected througha narrow passage. The sample and lysis buffer can be mixed bytransferring the sample and buffer mixture back and forth from onevessel to the other. One of the vessels can contain, for example, anextraction matrix for extracting nucleic acids from the sample andbuffer mixture. Repeated transfer of the mixture ensures thorough mixingand offers multiple opportunities for nucleic acids to be extracted fromthe mixture by the matrix.

The utility of many chromatography-based devices and procedures may belimited; such devices may not be suitable for performing chemicalsyntheses, high-throughput analyses, or processing sequences involvingmultiple buffers, solutions and/or reagent in an automated manner, andthe separated chemical species may be eluted in relatively large volumesof elution buffer, necessitating a concentration step before the elutedchemical species may be used for subsequent analysis or furthertreatment.

Many standard procedures exist for detection and analysis of one or moreparticular chemical species in a liquid sample. In many cases, suchprocesses require that the sample be subjected to certain preparatorysteps prior to the actual detection or analytical steps. In some cases,the sample preparation may be labor-intensive. Also, in some cases, thesample preparation may be incompatible with the detection or analyticalmethod, e.g., the pH of buffers used in sample preparation and theanalytical method may be different and incompatible. In such cases, itmay be necessary to manually adjust one or more chemical properties ofthe prepared sample (e.g., pH, concentration, etc.) prior to performingthe desired detection or analytical test. Consequently, efficiency ofthe overall process may be reduced, even to the point that the devicesand processes being used may become unsuited for high-throughputanalysis of the samples.

Therefore, a need exists for a device that may be employed in order tosubject a liquid sample to one or more of a broad range of chemicaltreatments. Furthermore, a need exists for such a device that may beautomated or is otherwise suitable for use in high-throughput analyses.

SUMMARY OF THE INVENTION

The present invention provides a device that is highly versatile and maybe used to subject a liquid sample to one or more of a wide variety ofchemical processing operations. Various embodiments of a deviceaccording to the present invention may provide the ability to performhigh-throughput chemical processing, multi-step chemical processing,analyte detections, sample preparation, chemical separations and thelike, as well as combinations of two or more of any of the foregoing.

Thus, the present invention provides a device for treating a liquidsample including: a first chamber having a variable volume; a secondchamber having a variable volume; a third chamber having a variablevolume; an interconnect comprising one or more passageways, eachpassageway engaged with one or more chambers so that the interconnectprovides fluid communication between the first chamber, second chamberand third chamber; and a treating substance included in at least one ofthe first chamber, the second chamber, the third chamber, or at leastone passageway of the interconnect; wherein the device is configured sothat movement of the liquid sample through the device is controlled bychanging the variable volumes of two or more chambers.

In certain embodiments, syringes or bladders may provide thevariable-volume chambers. The device may include additional elementssuch as one or more detection elements, temperature-regulation elementsor actuators for regulating the volumes of one or more chambers. Suchadditional elements may be controlled by a controlling element such aprogrammable microprocessor.

In another aspect, the present invention provides an apparatus forsubstantially simultaneously treating a plurality of liquid samples, theapparatus including: a plurality of units, each unit including a firstchamber having a variable volume, a second chamber having a variablevolume, a third chamber having a variable volume, an interconnectcomprising one or more passageways, each passageway engaged with one ormore chambers so that the interconnect provides fluid communicationbetween the chambers, and a treating substance included in at least oneof the first chamber, the second chamber, the third chamber or a portionof the interconnect; an actuator connected to two or more first chambersfor regulating the plurality of first chamber volumes; an actuatorconnected to two or more second chambers for regulating the plurality ofsecond chamber volumes; and an actuator connected to two or more thirdchambers for regulating the plurality of third chamber volumes.

Various other features and advantages of the present invention shouldbecome readily apparent with reference to the following detaileddescription, examples, claims and appended drawings. In several placesthroughout the specification, guidance is provided through lists ofexamples. In each instance, the recited list serves only as arepresentative group and should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plan view of a cartridge having a variable-volume chamberaccording to the present invention;

FIGS. 2A, 2B, and 2C are schematic drawings illustrating a singletreatment operation sequence utilizing a device having four chambers;

FIG. 3 is a schematic drawing illustrating an optional step that employsreversible flow;

FIG. 4 is a perspective view of one embodiment of a device according tothe present invention;

FIG. 5 is a plan cross-section view of a portion of the device depictedin FIG. 4; and

FIG. 6 is a perspective view of one embodiment of an apparatus forsupporting and actuating multiple treatment units.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE INVENTION

The fields of genomics and proteomics present challenges to modernchemical and biochemical processing systems. High-throughput, multi-stepprocessing may be desirable for detection and/or analysis ofnucleotides, in the case of genomics, or proteins, in the case ofproteomics. In one aspect, the present invention provides a devicesuitable for performing a wide range of chemical processes on a liquidsample. As used herein, liquid sample may refer to an untreated liquidsample, such as a liquid sample as it is initially loaded into thedevice. Alternatively, the term liquid sample may include anypartially-treated sample: either a) a sample that has been subjected tosomething less than an intended plurality of treatment steps in amulti-step treatment sequence, or b) that portion of a sample that hasbeen treated if, for example, only a fraction of the total sample volumeis subjected to a particular treatment while holding the remainder ofthe sample in either temporary or permanent reserve.

In another aspect, the present invention provides a system forperforming multiple chemical processes on a liquid sample, such asmultiple sample preparation process, detection process, analyticalprocess, or any combination of two or more such processes. In yetanother aspect, the present invention provides a system in which aplurality of liquid samples may be subjected to one or more chemicalprocess to achieve high-throughput utility. The samples may be treatedin parallel, substantially simultaneously, or both.

A device according to the present invention may be suitable for treatingone or more liquid samples by performing one or more chemical processeson the one or more liquid samples. Suitable chemical processes include,but are not limited to, mixing, reacting, filtering, dialyzing,synthesizing, fractionating, detecting, catalyzing (includingenzymatically catalyzing) reactions, performing various separations, andcombinations thereof.

The device generally includes a plurality of chambers having variablevolumes. As used herein, the term chamber may refer to any structuralform at least partially defining an internal volume. When used in thisway, the term chamber includes the structure, such as a syringe or abladder, that at least partially defines the internal volume. Thus,another element of the device may be attached to a portion of a chamber.Chamber also may be used to refer to the variable internal volume of,for example, a syringe, bladder, cartridge, or the like.

In some embodiments of the device of the present invention, chambers maybe reversibly removable from the device. Thus, sample may be more orless continuously supplied by replacing a used sample chamber, emptiedto any desired extent, with a replacement sample chamber that has beenfilled to a desired extent while the replacement chamber was off-line.

Some embodiments of the present invention may include disposableelements such as disposable syringes as variable-volume chambers.Devices having such disposable variable-volume chambers may reduce thecost of performing a chemical processing sequence that can employ suchinexpensive disposable elements.

The chambers are in fluid communication with one another through aninterconnect that provide fluid communication between the chambersthrough one or more passageways. The interconnect may be constructed ofany suitable material that can provide one or more conduits orpassageways between the chambers. The interconnect may be constructed ofinexpensive material that allows the interconnect to be disposable.Thus, in combination with disposable variable-volume chamber elements,the device of the present invention may include a series of modular,disposable elements that permit one to rapidly reconfigure the device toperform a different chemical process treatment.

The interconnect may include a single central bore through its center asa passageway. Alternatively, the interconnect may include a plurality ofpassageways. For example, an interconnect having two ends may includetwo distinct end-to-end passageways with the passageways in fluidcommunication at a first end, but being in fluid isolation from oneanother along their length and each passageway being in fluidcommunication with a different chamber at the second end. If a pluralityof chambers are present, the interconnect may be branched or unbranchedin order to provide any desired level of fluid communication between theplurality of chambers.

The versatility of the present invention is provided, in part, byselection of a treating substance. As used herein, a treating substanceis any substance that promotes chemical processing of a liquid sample,e.g., by one or more of the chemical processes identified above.Suitable treating substances include, but are not limited to, reactants,buffers, filters, membranes, beads, size exclusion media, functionalizedsolid supports, catalysts (including enzymes), markers, affinityreagents, and combinations thereof. A treating substance may be providedin any suitable form. For example, a treating substance may be providedas a filler, occupying at least a portion of a lumen in a passageway ora chamber. Alternatively, certain treating substances may be provided asa coating on a surface of a passageway or chamber.

A treating substance may be located in any portion of the device thatpermits contact with at least a portion of the liquid sample, therebyproviding the opportunity for the treating substance to perform thedesired treatment. Suitable locations for the treating substanceinclude, but are not limited to, any chamber, any portion of theinterconnect, including one or more passageways, and any combinationthereof.

Because the device includes a plurality of potential locations forplacement of the treating substance, it may be possible to design adevice that provides more than one treating substance. When two or moretreating substances are provided, they may be co-localized within thedevice or, alternatively, provided at two or more locations within thedevice. In one embodiment, for example, a functionalized support isprovided in the interconnect and an elution buffer is provided in one ofthe chambers. Thus, the device of the present invention may be designedto perform a plurality of chemical treatments on a single liquid sample.

In another example, one treating substance may be provided as a coatingon the lumen surface of a passageway and a second treating substance maybe provided as a filler, occupying a portion of the volume of thepassageway. Alternatively, a passageway may be filled or coated with amixture of two treating substances. Thus, a single sample may be treatedby each of the two treating substances substantially simultaneously. Inyet another example, a device having an interconnect with dualpassageways may include different treating substances in eachpassageway. In this way, one can perform two different chemicaltreatments on a single sample. The two treatments may be performed inparallel, substantially simultaneously, or both.

In an embodiment of the present invention in which the interconnectcomprises a plurality of passageways, as described above, eachpassageway may include a different treating substance than another ofthe plurality of passageways. For example, an interconnect having twodistinct end-to-end passageways with the passageways in fluidcommunication at a first end, but otherwise being in fluid isolationfrom one another and in fluid communication with different chambers atthe second end may include different treating substances in the twopassageways. When used by moving liquid from the first end to the secondend, such an interconnect may allow one to divide a sample and providetwo different chemical treatments to the divided sample. When used bymoving liquid from the second end to the first end, such an interconnectmay allow one to combine two solutions, such as reagents, that haveseparately received different chemical treatments. Alternatively, thesame treating substance may be provided in each passageway so that twosolutions can be combined that have separately received similar chemicaltreatments. In other embodiments, the interconnect may include three ormore passageways.

Another feature of the present invention, as will be explained in moredetail below, is that a liquid, such as a sample, buffer, reactant, orwash solution, may be directed through the device through thecoordinated control of the volumes of one or more chambers, so-called“passive valving.” Thus, the flow of liquid may be directed through thedevice in a particularized sequence merely by controlling the volumes ofselected chambers. In a device in which a plurality of treatingsubstances has been provided, a sample may be subjected to multiple,sequential treatments by selectively directing liquid through the devicein a desired sequence. A device according to the present invention mayinclude one or more check valves or other mechanically or electronicallycontrolled valves. However, passive valving is inexpensive and may proveless prone to failure.

Either the sample or the treating substance, if suitably liquid, may bethe liquid directed through device in order to perform multiple chemicaltreatments. That is, if a treating substance is liquid, it may bedirected to contact the sample at the appropriate time during thetreatment process. Alternatively, the sample may be directed to contactthe desired treating substance at the appropriate time during thetreatment process. A multi-step treatment sequence may use any suitablecombination of directing liquid sample, treating substances, mixturesthereof, or any combination of the foregoing in order to perform thedesired chemical processing. In general, the versatility provided by theability to control the flow of liquids through the device allows one todesign and/or select any desired processing sequence.

Another feature of the present invention is that the device may includeany number of chambers simultaneously connected to the interconnect.Thus, complex treatments that include multiple reactants, buffers,washes, and the like may be performed using the present invention,merely by designing the device to provide additional chambers that havebeen preloaded with materials desired for the complex treatmentsequence. In this way, multi-step chemical processing sequences may beperformed without having to remove and replace chambers in order toprovide treating substances for subsequent steps in a multi-stepprocessing sequence. This embodiment of the device of the presentinvention may be particularly suited to performing automated,high-throughput chemical process sequences such as those desirable forsample preparation for genomic or proteomic analyses.

In some embodiments, however, the chambers may be reversiblyinterchangeable in the device. That is, the device may includereversibly sealable ports, through which fluid communication between onechamber and the remainder of the device can be established. Thus, thecontents of a chamber, e.g., a wash buffer, may be dispensed from thechamber into the remainder of the device to the desired extent. The usedchamber may be removed and replaced with another chamber containingcontents desired for a subsequent step in the process sequence, e.g., areactant or another wash buffer.

A device according to the present invention also may include one or moreadditional elements selected to facilitate particular steps in achemical treatment process. For example, a device may include one ormore detection elements designed to detect at least a portion of atreated or untreated sample. Suitable detection elements include, butare not limited to, spectrophotometric detectors, fluorescencedetectors, pH detectors, electrical conductivity detectors or refractiveindex detectors. Such detector elements may be integrally incorporatedinto the device or may be an element external to the device itself, butadapted for use with the device. Detector elements may be adapted todetect one or more chemical species in the interconnect or one or morechambers.

Another example of an additional element that may be used in connectionwith a device according to the present invention is atemperature-regulating element. Such an element may regulate thetemperature in the interconnect or one or more chambers in order to atleast partially control the progress of a particular step in a chemicalprocessing sequence. For example, the kinetics of certain types ofreactions or other chemical interactions may be controlled, at least inpart, by controlling the temperature at which such steps are performed.

Additionally, actuators may be attached to the chambers for controllingthe variable volumes of the chambers, as will be described in greaterdetail below.

Detection elements, temperature-regulating elements and actuators may becontrolled by a controlling element, e.g., a programmablemicroprocessor. Controlling such elements with a controlling elementsuch as a programmable microprocessor allows one to design complexsequences of chemical processing steps, load the desired samples,buffers, reactants and the like, activate the controlling element andhave the sequence performed automatically, thereby reducing the manualcost of performing the chemical processing sequence.

The apparatus according to the present invention includes chambershaving a variable volume. Variable-volume chambers may be provided inany suitable mechanical form including, but not limited to, apiston-like device such as a syringe, a container with a flexible wallsuch as a bladder, an enclosure having an elastic element such as abellows, and a rolling diaphragm device. Some embodiments of the presentinvention can be constructed so that the variable-volume chambers areprovided by commercially available disposable syringes. Alternatively, avariable-volume chamber may be provided by a cartridge having an openend and a chamber defined, in part, by a chamber wall. The cartridge canserve as a syringe when equipped with a moveable plug fitted to form aslidable fluid-tight seal with the chamber wall when the plug isinserted into the cartridge through the open end.

FIG. 1 shows a plan view of a cartridge 20 suitable for providing avariable-volume chamber in connection with the present invention. Thecartridge 20 may include a cartridge body 24 that includes an open end26 and a septum 22 at the end of the cartridge body 24 generally opposedto the open end 26. A movable plug 28 may be disposed within thecartridge 20, thereby at least partially defining the variable-volumechamber 30. In certain embodiments, the walls of the cartridge body 24and the septum 22 may remain substantially stationary while the deviceis in use. In such embodiments, the variable nature of thevariable-volume chamber 30 is substantially dependent upon position ofthe moveable plug 28 within the cartridge 20.

The movable plug 28 may include a releasable connection 32 such asthreaded stud for convenient connection to means for controlling theposition of the moveable plug 28 and, therefore, the volume of thevariable-volume chamber 30. As just one example, a plunger rod may beconnected to the moveable plug 28, thereby adapting the cartridge formanual or automated control of the position of the moveable plug 28. Anymeans for controlling the position of the moveable plug 28 may besuitable for use in the present invention, however.

The cartridge 20, when used to provide the variable-volume chamber, alsomay include a lubricant to facilitate movement of the movable plug 28against the interior wall of the cartridge body 24. The lubricant may bean integral component of, or coated onto, at least a portion of themoveable plug 28, at least a portion of the cartridge body 24, or both.Suitable lubricants may be selected to be inert with respect tomaterials intended to be contained within the variable volume 30 of thecartridge 20. Suitable lubricants include, but are not limited to,silicones, silanes and hydrocarbons. In some embodiments, the septum 22may be fixed to the cartridge body 24 by a cap such as an aluminum cap34.

The present invention is hereafter described in terms of a device havingvariable-volume chambers provided by cartridges such as the cartridgeillustrated in FIG. 1 and described above. However, other mechanicalforms of providing a variable-volume chamber (such as a bladder,bellows, alternative forms of syringes, etc.) may be equally suitablefor use in the present invention, unless otherwise specified.

Also, identification of particular chambers as intended for loading of asample, reagent, buffer or the like, or for use as a collection chamber,is for convenience and illustrative purposes only. A feature of thedevice of the present invention is that the particular function of anyone chamber may be determined, in part, by the treatment operationsequence being followed by the operator.

FIGS. 2A, 2B, and 2C provide a schematic illustration of one example ofa treatment operation sequence using a device 100 that includes fourcartridges 101, 102, 103 and 104, each cartridge serving as a chamberhaving a variable volume. Such a treatment operation sequence might becharacterized as a single-pass sequence protocol because the liquidsample is contacted with a treating substance only once, i.e., in asingle pass. An interconnect 105 provides fluid communication betweenthe cartridges through an internal passageway (not shown). Eachcartridge may be engaged with the interconnect 105 so that such fluidcommunication between the chambers is maintained.

As one example of the single-pass sequence protocol (FIG. 2A), thevariable-volume chamber 131 of cartridge 101 may be filled with a liquidsample and, therefore, be designated the sample chamber. Thevariable-volume chamber 134 of cartridge 104 may be filled with a secondsolution, e.g., a wash buffer. Cartridge 102 and cartridge 103 can beginthe protocol with their movable plugs 117 and 118, respectively,positioned forward as shown, thereby minimizing the volume of theirrespective chambers 132 and 133. The interconnect 105 may contain atreating substance 160 such as an immobilized molecule selected tointeract with the liquid sample in a predetermined way, therebyproviding chemical treatment of the liquid sample.

FIG. 2B illustrates the movement of the liquid sample during asingle-pass protocol. The plunger rods of selected cartridges may beimmobilized to prevent movement of liquid into the chambers of theselected cartridges (e.g., plunger rods 114 and 113 are immobilized inFIG. 2B). The plunger rod 111, shown connected to the moveable plug 116in cartridge 101, may be advanced, thereby decreasing the volume ofchamber 131. The plunger rod 111 may be completely advanced, as shown inFIG. 2B, or only partially advanced, as desired. The rate and degree ofplunger rod advancement may be controlled manually or automatically.

As plunger rod 111 is advanced, the volume of chamber 131 is decreased,thereby forcing liquid sample out of chamber 131, into the interconnect105 and into contact with the treating substance 160. At the same time,plunger rod 112 and moveable plug 117 are displaced outward with respectto cartridge 102, thereby increasing the volume of chamber 132. Thus,the portion of the liquid sample that has been passed through theinterconnect 105 and, therefore, has been chemically treated by thetreating substance 160, is directed toward chamber 132. Plunger rod 111may be advanced until the desired amount of liquid sample has beendirected through the treating substance 160.

The outward movement of plunger rod 112 may be passive, i.e., it may bepushed outward by the treated sample being directed toward chamber 132as a result of decreasing the volume of chamber 131 while the plungerrods 113 and 114 remain immobilized. Alternatively, the outward movementof the plunger rod 112 may be active, i.e., the plunger rod 112 may bepulled outward. Such active outward movement of the plunger rod 112 maybe controlled manually or automatically.

If desired, one or more of the variable-volume chambers may be designedto minimize any residual volume 135 that may exist when the moveableplug has been advanced completely. Such minimizing of the residualvolume may increase the efficiency with which the liquid sample is used.

FIG. 2C illustrates a subsequent processing step executed after theliquid sample is dispensed from chamber 131 to the desired extent. Thedescription that follows is provided in the context of the subsequentchemical processing step being a wash step. However, any suitable stepin a chemical processing treatment operation sequence, e.g., an elutionstep, may be performed. Accordingly, any solution suitable for thedesired chemical processing step may be provided in chamber 134.

For a wash step, a wash solution may be provided in the variable-volumechamber 134 of cartridge 104. The plunger rod 111 of cartridge 101 maybe immobilized to prevent movement of liquid back into chamber 131. Theplunger rod 114 and moveable plug 119 are advanced, thereby directingwash solution into the interconnect 105. As described above for theadvancement of plunger rod 111, advancement of plunger rod 114, may becontrolled manually or automatically. If plunger rod 113 remainsimmobilized and plunger rod 112 remains moveable while plunger rod 114is advanced, then the wash solution entering the interconnect 105 fromchamber 134 will direct additional treated sample into chamber 132.

When the desired amount of treated sample is collected in chamber 132,plunger rod 112 may be immobilized and plunger rod 113 may be released,thereby allowing plunger rod 113 to be outwardly displaced with respectto cartridge 103 and increasing the volume of chamber 133. Anysubsequent advance of plunger rod 114 will cause treated sample, washsolution, or a mixture of both to be directed into chamber 133. Theoutward movement of the plunger rod 113 may be passive or active, asdescribed above for the control of the outward movement of plunger rod112.

As indicated above, the solution loaded in chamber 134 may provide oneor more functions in addition to, or in lieu of, providing a wash. Forexample, the solution loaded into chamber 134 may, for example,regenerate the treating substance 160 for treatment of a subsequentsample, or release a component that has been removed from the liquidsample by, for example, adsorption to the treating substance 160. In thelatter case, the component of the liquid sample released from thetreating substance 160 may be collected in the solution that fillschamber 133. In the treatment operation sequence illustrated in FIGS.2A, 2B and 2C, chamber 132 and chamber 133 contain processed solutionsthat have passed through interconnect 105, one or both of which may beuseful for subsequent analysis, detection or further treatment.

FIG. 3 provides a schematic illustration of an optional feature of atreatment operation sequence such as that shown in FIGS. 2A, 2B and 2C.This optional feature allows the device to perform a multi-pass sequenceprotocol in which the liquid sample is contacted with the treatingsubstance in multiple passes. FIG. 3 shows that the movement of theliquid between any two chambers may be reversible. The device isillustrated schematically in FIG. 3 as in FIGS. 2A and 2B. Movement ofthe liquid sample from chamber 131 to chamber 132 may be accomplished asdescribed above in the single-pass sequence protocol. After the liquidsample has been directed toward chamber 132 to the desired extent, thesolution flow may be reversed by directing flow from chamber 132,through the interconnect 105, and back toward chamber 131. The reversedflow may be accomplished by advancing the plunger rod 112 connected tothe moveable plug 117 into cartridge 102 while immobilizing the plungerrods 113 and 114 of cartridges 103 and 104, respectively. Again, theadvancement of plunger rod 112 may be controlled manually or actively.Furthermore, the outward movement of moveable plug 116 may be controlledpassively or actively, either manually or automatically, as describedabove.

Reciprocating the flow between chambers 131 and 132 may provide certainadvantages over a single pass through the interconnect 105 and treatingsubstance 160. For example, a single pass sometimes may not besufficient to treat the liquid sample to the desired extent. The cycleillustrated in FIG. 3 can be performed multiple times until the liquidsample has been treated to the desired extent.

Although illustrated and discussed with respect to chambers 131 and 132,flow between any two chambers may be reversed if desired for aparticular treatment operation sequence. Also, treatment sequences maybe designed that incorporate a plurality of such reversible-flow steps,if desired.

The treated sample may be collected in any suitable variable-volumechamber, e.g., chamber 132. In some embodiments, the treated sample maybe the desired substance. In other embodiments, one or more additionaltreatments steps may be desired to obtain the desired substance. Forexample, as described above with regard to the single-pass sequenceprotocol illustrated in FIGS. 2A-2C, a subsequent processing step suchas a wash step or an elution step may be performed in order to obtainthe desired substance. In one such protocol, the solution loaded inchamber 134 may release one or more desired molecules from the treatingsubstance 160 and be collected in a suitable variable-volume chamber,e.g., chamber 133.

More complex treatment operation sequences also may be performed using adevice according to the present invention. For example, a sample may befiltered by passing the sample (single- or multi-pass) through aselective filtration membrane as a treating substance, e.g., in theinterconnect, in one direction. The filtered material may be collectedby directing a wash solution in the opposite direction. The filtrate,filtered material, or both may be collected for further treatment,analysis or detection.

Also, synthetic reactions may be performed using a device according tothe present invention. Reactants may be provided in one or more chambersas gasses, solids or liquids. Reactants may be provided individually orcombined with at least one other reactant, for example, in solution, insuspension, in an emulsion, and the like. Moreover, molecules may besynthesized using a multi-step syntheses by merely providing eithersufficient numbers of chambers to provide all of the required reactantsor replacing used chambers with chambers loaded for use in a subsequentstep of the synthesis. Such a complex synthetic pathway may include oneor more clean-up steps such as filtration or separation in order toseparate reaction products. One or more temperature-regulating elementsmay be employed to control the temperature at one or more steps in thesynthetic pathway.

As another example, a device according to the present invention may beused to fractionate a sample. If many fractions are desired, continuousfractionation may be accomplished using a plurality of reversiblyremovable chambers for collecting the fractions. For example, the samplemay be fractionated continuously by alternating the flow of the samplebetween two collection points while sequentially filling, removing andreplacing the collection chambers connected to the treatment unit ateach collection point. In this way, the flow of sample may be controlledso that the flow can be diverted when a particular collection chamber isfilled to the desired extent, thereby allowing fraction collection tocontinue while the filled collection chamber is removed and replacedwith a new collection chamber.

Complex chemical sequences that incorporate one or more different kindsof processes such as those described above may be performed using adevice according to the present invention. Furthermore, because such adevice may be controlled by a controlling element, such as aprogrammable microprocessor, such complex sequences may be performedunder automated control, if desired.

FIG. 4 provides a perspective exploded view of one embodiment of adevice 100 suitable for carrying out the processes just described. Theillustrated embodiment of the device 100 includes a plurality ofvariable-volume chambers, provided by four cartridges 101, 102, 103 and104 and four plunger rods 111, 112, 113 and 114. An interconnect 105provides fluid communication between the variable-volume chambers. Inthis example the interconnect 105 is in the shape of a “U” so that allof the plunger rods may be controlled from one side of the device. The“U”-shaped interconnect 105 permits one to easily control the positionof plunger rods and, therefore, the volume of chambers on opposite sidesof the interconnect because the ends of plunger rods controlling thevolumes of the opposed chambers may be positioned side-by-side. This maybe particularly advantageous if the plunger rods are being controlledmanually because the operator can control the volumes of opposedchambers without reaching across the length of the device.

Cartridges 101 and 104 may be designed to fit within a housing 200 whilecartridges 102 and 103 may be designed to fit within a second housing202. Plunger rods 111, 112, 113 and 114 may be sized and configured tobe received in the open ends 126 of cartridges 101, 102, 103 and 104,respectively. Each plunger rod may be equipped at one end with threads204 adapted to receive a thread stud 136 anchored in a movable plug 128,as shown in FIG. 5. Each plunger rod also may have an end 206 adapted toengage an automatic control device that will be described in more detailbelow. Alternatively, if the device designed to be operated under manualcontrol, then thumb plates may be provided on the end of each plungerrod.

The device 100, as depicted in FIG. 4, may include retainers 208 and 210that may serve to protect and support the cartridges, particularly whenthe cartridges may be susceptible to damage, such as when constructedfrom glass or when the cartridges are sufficiently longer than thehousings 200 and 202 that additional support may be desirable. Theretainers 208 and 210, when present, may be sized and shaped to receiveone or more cartridges. The retainers 208 and 210 may include attachmentmeans for releasably engaging the housings 200 and 202, respectively. Inone embodiment, the attachment means are manually releasable and mayreattach the retainers to the housings. The retainers may, for example,connect to the housings by a press fit or, alternatively, have anextending arm 212 that latches over a lip 214 near the proximal end ofthe housing 200.

Each of the housings 200 and 202 may include a nozzle 220. Inembodiments in which the housing can accommodate a plurality ofcartridges, the nozzle 220 may include a plurality of openings. Forexample, the embodiment illustrated in FIG. 4 includes a housing thatcan accommodate two cartridges and a nozzle 220 that has two openings222 and 224. This may be seen with more particularity in connection withthe embodiment illustrated in FIG. 5; opening 222 leads to chamber 131and opening 224 leads to chamber 134.

The interconnect 105 may include a first end adapter 230 and a secondend adapter 232, each configured to engage the nozzle end of thehousing. In one embodiment, each of the end adapters 230 and 232includes a base flange 234 adapted to engage complementary grips 236 and238 adjacent to nozzle 220. However, any means for providing afluid-tight seal between the interconnect 105 and the chambers may besuitable.

FIG. 5 provides a cross-section plan view of a portion of one embodimentof the device 100 according to the present invention. In thisembodiment, the housing 200 includes a pair of piercers 240, eachpiercer configured to penetrate a septum 22 of a cartridge, therebyproviding fluid communication between the chamber within each cartridgeand the corresponding opening (222 or 224) of the nozzle. Each of thepiercers 240 includes a hollow needle 246 anchored in a piercer body242. The hollow needle 246 provides fluid communication between thechamber of a cartridge and a plenum 248. Therefore, for example, chamber131 is in fluid communication with the interconnect 105 via needle 246,plenum 248 and opening 222. An O-ring 256 may be seated around thepiercer bodies 242 to provide a fluid-tight seal for plenum 248. Afinger flange 260 may be provided to facilitate manual actuation of thedevice.

A device according to the present invention may be designed to standalone. Alternatively, multiple devices may be incorporated as treatmentunits 400 in a larger apparatus 300, as shown in FIG. 6. The apparatus300 may be designed for supporting and actuating multiple treatmentunits 400, thereby allowing chemical processing of multiple liquidsamples in a single apparatus 300 to be performed in parallel. Theapparatus 300 may be further designed to permit the chemical processingof multiple samples to be performed substantially simultaneously. Theapparatus depicted in FIG. 6 provides for six treatment units 400 to besupported and actuated, but an apparatus 300 may be designed toaccommodate any desired number of treatment units 400.

Housing support blocks 304 may be stacked on a base 302. The housingssupport blocks 304 may have recesses formed therein that are shaped sothat each block can receive and support securely one or more treatmentunits 400. The support blocks 304 may be constructed of any suitablesupportive medium including, but not limited to, polystyrene,polytetrafluoroethylene, polycarbonate, polypropylene and the like. Therecesses may be formed by any process suitable for the particularmaterial used to form the support blocks 304. For example, the recessesmay be molded or milled into the support blocks 304.

If present, detection elements or temperature-regulating elements may beincorporated into or attached to the support blocks 304.

During operation of the apparatus 300, the support blocks 304 may besecured by any suitable means, e.g., a capture plate 306, as shown inFIG. 6, which may be bolted to one or more structural members 310 withbolts 308.

In certain embodiments, multiple treatment units 400 may be supportedwithin a rack of support blocks 304 such that the treatment units 400may be easily and quickly engaged or disengaged from the rack. Also, thetreatment units 400 may be designed so that the element having thevariable volume chamber, e.g., a syringe or bladder, may be easily andquickly engaged or disengaged from the treatment unit 400.Alternatively, the rack of support blocks may be easily and quicklyengaged or disengaged from the base 302.

Embodiments in which at least a portion of the apparatus 300 may beeasily and quickly engaged or disengaged may be particularly useful forcertain applications. For example, such designs may permit one tocollect fractions of a treated sample in separate chambers, e.g.,syringes, and insert replacement collection chambers into the apparatus300 for continuous, uninterrupted collection of treated sample.

In some embodiments of the apparatus of the present invention, aplurality of actuators may be present. The actuators, when present, maybe designed for control of plunger rods associated with the treatmentunits. The actuators may be configured to provide any desired level ofcoordinated control of the plunger rods of one or more treatment units.Such coordinated control of plunger rods from multiple treatment unitspermits one to control the movement of liquid within multiple treatmentunits simply by controlling a single actuator. Thus, one may control theprogress of multiple chemical processing sequences substantiallysimultaneously.

The embodiment depicted in FIG. 6 includes four actuators 321, 322, 323and 324, each actuator designed to control the volumes of analogouschambers in each treatment unit housed within the apparatus. Becauseeach treatment unit may include any number of variable volume chambersand each variable volume chamber may or may not be connected to anactuator, an apparatus 300 according to the present invention mayinclude any number of actuators.

If coordinated control of chemical processing of multiple samples inparallel is desired, each actuator may be adapted to engage analogousplunger rods of each treatment unit. For example, actuator 321 may beengaged with the plunger rod 111 of the sample chamber of each treatmentunit, the actuator 322 may be engaged with the plunger rod of a firstcollection chamber of each device, and so on. As shown in FIG. 6, eachactuator may include multiple receptacles 326 for releasably connectingto plunger rods. The receptacles 326 may include quick release fastenercomplementary to a fastening structure on the plunger rod, e.g., abutton end 206.

Each actuator is reversibly slidable, thereby capable of controlling themovement and location of each plunger rod attached to the actuator.Because the location of the moveable plug, which is attached to theplunger rod, at least partially determines the volume of the variablevolume chamber that at least in part directs the flow of liquid withinthe treatment unit, the actuators may be used for parallel control themovement of liquid in each of a plurality of treatment units.

During the reversible sliding motion, the actuators may be supported byslide rods 328 and driven by, for example, lead screws 330. Othermethods of creating controlled linear motion are possible. The leadscrews 330 may be driven by motor-turned gears within a drive unit 340.Drive unit 340 also may include a control mechanism for the timing ofthe various motions that are needed for the different procedures,thereby providing automated control of the chemical processing sequencebeing performed in each treatment unit 400 of the apparatus 300. Suchautomated control may be particularly useful for performinghigh-throughput processing sequences such as sample preparation forgenomic or proteomic analyses. The control mechanism may include amicroprocessor, although alternative control mechanisms, such as camfollowers or relay controllers, may be suitable.

Each actuator may be connected to the control mechanism so that thecontrol mechanism can control the movement of each actuator. In thisway, the volumes of the various chambers of each treatment unit 400 inthe apparatus 300 are controlled by the control mechanism, thereby alsocontrolling the movement of liquids from one chamber in a treatment unitto other chambers within the same treatment unit. Also, because eachactuator may be connected to multiple analogous plunger rods,controlling the position of multiple analogous moveable plugs and,therefore, the volumes of multiple analogous chambers, the controlmechanism can control the parallel processing of multiple samplessimultaneously.

For simplicity, various features of the present invention have beendescribed in isolation. Such descriptions shall not be construed tolimit the scope of the present invention. One of the features of thepresent invention is the versatility of design provided by the deviceand the broad variety of chemical treatments that may be performedaccording to the present invention. Thus, the features of certainparticular embodiments may be combined with the features of otherembodiments to obtain additional embodiments of the present invention.

EXAMPLES

The following examples have been selected merely to further illustratefeatures, advantages, and other details of the invention. It is to beexpressly understood, however, that while the examples serve thispurpose, the particular materials and amounts used as well as otherconditions and details are not to be construed in a matter that wouldunduly limit the scope of this invention.

In each example, glass cartridges (80 mm length and 7 mm internaldiameter available from Kimble, Vineland N.J.) were used to containliquid solutions or solids, as appropriate. The glass cartridges eachincluded an open end and a nubbed end generally opposed to the open end.The cartridges were silicone-treated (560 Medical Fluid available fromDow Corning, Midland, Mich.) according to the manufacturer'srecommendation to provide a lubricated surface to facilitate plungermovement within the cartridges.

A moveable rubber plug (7 mm diameter) having a threaded metal post(available from Abbott, Abbott Park, Ill.) was inserted into the openend of each glass cartridge. Each threaded post was attached to aplunger so that the position of the plug could be externally controlled.

Some cartridges were loaded with a solution or solid, as appropriate.Solution or solid was introduced into an opening at the nubbed end ofthe cartridge. The rubber plug was advanced until the solution or solidwas level with the opening at the nubbed end of the cartridge. Thenubbed end of the cartridge was crimp-sealed using a rubber-linedaluminum septum cap (available from Wheaton Pharmatech, Salisbury, Md.).

Other cartridges were intended for collection of treated samples.Collection cartridges were assembled by advancing the plug to its mostforward position in the cartridge. The nubbed end of the cartridge wassealed using a rubber-lined aluminum septum cap as described above.

In each of the following examples, a dual barrel syringe housing(described in applicants' copending International Publication No. WO01/67961, filed Mar. 10, 2000) was fitted with two glass cartridgesassembled as described above and attached to each end of a glass column,thereby forming a closed system having four syringe chambers (two oneach side of the glass column). The glass column (100 mm length and 3 mminternal diameter, available from Omnifit, Cambridge, England) waseither packed with packing materials or used empty as a fluid conduit.When filled with packing material the column was sealed with endfittings containing 25 μm frits (also available from Omnifit). Each endof the column was attached to a dual barrel syringe housing withLuer-lock fittings.

In each example, cartridges (containing reagents, washes, buffers orempty, as appropriate for the particular example) were loaded into thedual barrel syringe housings. Each syringe housing included an inserthaving two 16-gauge needles capable of piercing the rubber-lined septumcaps of the glass cartridges to be fitted into the housing. Each needleprovided a conduit between a cartridge and an opening in a nozzle of thesyringe housing. Each needle and its associated nozzle opening providedfluid communication between a cartridge loaded in the housing and thecolumn.

Fluid movement through the system was controlled by immobilizing plungerrods associated with cartridges that were neither sources nordestinations of the liquids being transferred in a particular step.Positive pressure was placed on the plunger rod of the cartridge orcartridges that contained liquid to be transferred at a particular time.The plunger rod of a collection cartridge, i.e., the destination of theliquids being transferred during a particular step, was passivelyallowed to slide within its cartridge, thereby allowing the volume ofthe chamber in the cartridge to increase in response to the positivepressure applied to the source cartridge plunger rod(s). Unlessotherwise indicated, liquid was directed through the system at a rate ofapproximately 0.5 mL/min.

Example 1 Ionic Immobilization

The system described above was used to construct an ionic immobilizationsystem that was used for each of two runs, a single-pass run and amulti-pass run. For each run, the glass column was packed with 4% beadedagarose with immobilized iminodiacetic acid (available from Pierce,Rockford, Ill., copper ion binding capacity >0.9 mg). The column wasequilibrated with distilled and deionized water to remove sodium azidepreservative. Copper sulfate was dissolved in distilled and deionizedwater to form a 165 mM solution. 1.5 mL of the solution was loaded intoa first glass cartridge. 2.5 mL distilled and deionized water was loadedinto a second glass cartridge.

The first and second cartridges were inserted into one dual barrelsyringe housing. A third cartridge and fourth cartridge were insertedinto another dual barrel syringe housing attached to the other end ofthe glass column. The third and fourth cartridges were empty wheninserted into the housing.

In the single-pass run, positive pressure was applied to the plunger rodof the first cartridge, thereby introducing the copper sulfate solutioninto the glass column and contacting the copper sulfate solution withthe beaded agarose packed therein. The plunger rods of the second andfourth cartridges were immobilized, but the plunger rod of the thirdcartridge was not immobilized. Thus, after contacting the beadedagarose, the treated sample was directed toward and collected in thechamber of the third cartridge. When the plunger rod of the firstcartridge was fully advanced, all of the copper sulfate had beenintroduced into the column.

The column was washed by immobilizing the plunger rod of the firstcartridge and applying positive pressure to the plunger rod of thesecond cartridge so that the distilled water loaded in the secondcartridge was introduced into the column, thereby washing unbound copperions from the column. Because the plunger rod of the third cartridge wasnot immobilized, unbound copper ions washed from the column weredirected toward and collected in the third cartridge. Thus, all of theunbound copper ions were collected in the third cartridge.

In the multi-pass run, the plunger rods of the second and fourthcartridges were immobilized as at the beginning of the single-pass rundescribed above. Positive pressure was alternately applied to theplunger rods of the first and third cartridges so that the coppersulfate solution was passed back and forth through the agarose-packedcolumn nine times. Whenever positive pressure was being applied to theplunger rod of one of the first or third cartridges, the plunger rod ofthe other cartridge (“the passive cartridge”) was allowed to bedisplaced outward with respect to the cartridge, thereby allowing thesolution to temporarily collect in the passive cartridge.

After nine passes through the column, the solution was collected in thethird cartridge. Thereafter, the column was washed as described in thesingle-pass run. Thus, all of the unbound copper ions were collected inthe third cartridge.

Approximately 3.8 mL of treated liquid was collected in the thirdcartridge in each run. The amount of copper ion in the startingsolution, collected in the single-pass run and collected in themulti-pass run were analyzed spectrocolorometrically at 650 nm. Thestarting solution in each run contained approximately 15.73 mg of copperion, far in excess of the binding capacity of the column.

The collected solution from the single-pass run contained 15.70 mgcopper ions, indicating that the column immobilized 0.03 mg of copperion in the single-pass run. The collected solution from the multi-passrun contained 15.43 mg of copper ions, indicating that the columnimmobilized 1.3 mg of copper ion in the multi-pass run. Thus, the columnbound copper ions to less than 3% of capacity using the single-passprotocol, but the column bound copper ions to substantial saturationusing the multi-pass protocol.

Example 2 Affinity Immobilization

An affinity immobilization system was constructed and used for each oftwo runs, a single-pass run and a multi-pass run. The system wasconstructed as described in Example 1, with the following exceptions:the glass column was packed with Mimetic Blue SA P6XL beads (availablefrom ProMetric BioSciences Ltd., Isles of Man, British Isles) and wasequilibrated with 25 mM sodium phosphate buffer, pH 5.5; 2.0 mL of humanserum albumin was loaded into a first glass cartridge; and a wash buffercontaining 2.5 mL 25 mM sodium phosphate, pH 5.5 was loaded into asecond glass cartridge.

The human serum albumin solution was prepared by dissolving human serumalbumin (HSA available from Sigma Chemical Co., St. Louis, Mo.) in 25 mMsodium phosphate buffer, pH 5.5 to a concentration of 8.45 mg/mL.

The first and second cartridges were inserted into one dual barrelsyringe housing. A third cartridge and fourth cartridge were insertedinto another dual barrel syringe housing attached to the other end ofthe glass column. The third and fourth cartridges were empty wheninserted into the housing.

The single-pass run was performed in the same manner as the single-passrun of Example 1. Thus, all of the unbound HSA was collected in thethird cartridge.

The multi-pass run was performed in the same manner as the multi-passrun of Example 1. Thus, all of the unbound HSA was collected in thethird cartridge.

Approximately 4.3 mL of treated solution was collected in each run. Theamount of HSA loaded into each first cartridge and collected in eachthird cartridge was determined using the biocinchoninic acid (BCA)protein assay (available from Pierce, Rockford, Ill.). Approximately16.9 mg HSA was loaded into each first cartridge. 3.6 mg of unbound HSAwas measured in the collected solution in the single-pass run,indicating that approximately 13.3 mg of HSA was bound to the column inthe single-pass run. 3.3 mg of HSA remained unbound in the multi-passrun, indicating that approximately 13.6 mg of HSA was bound to thecolumn in the multi-pass run.

Example 3 Hydrophobic Immobilization and Elution

A hydrophobic immobilization system was constructed and used for each oftwo runs, a single-pass run and a multi-pass run. The system wasconstructed as described in Example 1, with the following exceptions:the glass column was packed with dodecyl agarose (available from Sigma,St. Louis, Mo.) and was equilibrated with phosphate buffered saline(PBS), pH 7.2; a peptide solution was loaded into the first cartridge;and a wash buffer containing 2.5 mL PBS at pH 7.2 was loaded into asecond cartridge.

The peptide solution was prepared by proteolyzing bovine serum albumin(BSA available from Sigma, St. Louis, Mo.) that had been dissolved inPBS, pH 7.2. Proteolysis was conducted by adding 1.6 mg TCPK trypsin(available from Pierce, Rockford, Ill.) that was freshly dissolved inwater. The proteolysis solution was allowed to incubate for 72 hours at37° C. The resulting peptide solution contained peptides at aconcentration of 304 mg/mL, based upon spectrophotometric analysis at214 nm.

The first and second cartridges were inserted into one dual barrelsyringe housing. A third cartridge and fourth cartridge were insertedinto another dual barrel syringe housing attached to the other end ofthe glass column. The third and fourth cartridges were empty wheninserted into the housing.

The single-pass run was performed in the same manner as the single-passrun of Example 1. Thus all unbound peptide was collected in the thirdcartridge.

The multi-pass was performed in the same manner as the multi-pass run ofExample 1. Thus, all unbound peptide was collected in the thirdcartridge.

Approximately 3.5 mL of solution was collected in each run. The amountof BSA peptides in the original sample solution and each collectedsolution was determined by spectrophotometric analysis at 214 nm. Thestarting solution in each run contained approximately 456 mg (1.5 mL) ofBSA peptides. 240 mg of unbound peptides were collected in theflow-through of the single-pass run, indicating that 216 mg of BSApeptides were immobilized by the column. 247 mg of unbound peptides werecollected in the flow-through of the multi-pass run, indicating that 209mg of BSA peptides were immobilized in the multi-pass experiment. Thus,there was no discernable increase in hydrophobic immobilization of BSApeptides by increasing the number of passes through the dodecyl agarosecolumn.

Next, the immobilized peptides were eluted from each of the single-passrun column and the multi-pass run column. For each elution, the firstand second cartridges were replaced in one syringe housing withcartridges containing 2.5 mL of 70/30 acetonitrile/water (v/v)containing 0.1% trifluoroacetic acid as an elution buffer. Also, thethird and fourth cartridges were replaced with empty collectioncartridges in the other dual barrel syringe housing.

Each elution was performed in the same manner as the single-pass run ofExample 1. Thus, in each elution, protein eluted from the column wascollected in the third cartridge.

The volume of collected eluate was 4.7 mL for each elution. The amountof eluted BSA peptide collected in the third cartridge of each elutionwas determined by measuring the absorbance at 214 nm. The mass of BSApeptides eluted from the single-pass run was 110 mg (of the 216 mghydrophobically bound to the column in a single pass. The mass of BSApeptides eluted from the multi-pass run was 128 mg (of the 209 mg of BSApeptides immobilized to the column in he multi-pass run).

Example 4 Size Exclusion Separation

A size exclusion separation system was constructed as described inExample 1, with the following exceptions: the glass column was packedwith Sephadex G-25 (available from Amersham Pharmacia Biotech AB,Uppsala, Sweden) and was equilibrated with phosphate buffered saline(PBS), pH 7.2; 200 μL of a dye-labeled protein solution was loaded intoa first glass cartridge; and a wash buffer containing 2.5 mL PBS buffersolution at pH 7.2 was loaded into a second glass cartridge.

The dye-labeled protein solution was prepared by dissolving 4 mg ofrhodamine-labeled BSA (Albumin, bovine-sulforhodamine 101 acid chloride,available from Sigma, St. Louis, Mo.) in 200 μL PBS, pH 7.2.

The first and second cartridges were inserted into one dual barrelsyringe housing. A third cartridge and fourth cartridge were insertedinto another dual barrel syringe housing attached to the other end ofthe glass column. The third and fourth cartridges were empty wheninserted into the housing.

The plunger rods of the second and fourth cartridges were immobilized,but the plunger rod of the third cartridge was not immobilized. Positivepressure was applied to the plunger rod of the first cartridge, therebycausing the dye-labeled protein solution to flow into the bead-packedcolumn at a rate of 0.5 mL/min. The labeled BSA solution formed a purplelayer on the bead bed.

The plunger rod of the first cartridge was immobilized and the plungerrod of the second cartridge was released from immobilization. Positivepressure was applied to the plunger rod of the second cartridge, therebycausing wash solution to flow into the column at a rate of 0.1 mL/min.

Separation of the rhodamine-labeled BSA from unreacted rhodamine wasobservable on the column during the flow through. The dye-proteinfraction was contained in a first clearly delineated band. Unreacted dyemolecules were contained in a trailing clearly delineated band. Thus,the dye-protein band was collected in the third cartridge. When theentire dye-protein band had been collected, the plunger rod of the thirdcartridge was immobilized and the plunger rod of the fourth cartridgewas released from immobilization. As positive pressure continued to beapplied to the plunger rod of the second cartridge, the unreacted dyeband was collected in the fourth cartridge.

Example 5 Cartridge Chemical Reaction

This example demonstrates the utility of the multiple cartridge designfor conducting chemical reactions. A system similar to that described inExample 1 was employed, with the following exceptions: the glass columnwas replaced with plastic tubing filled with 0.2 M sodium carbonatebuffer, pH 9.97; the first cartridge was loaded with 3.2 mg BSA solid;the second cartridge was loaded with FLORORLINK (Amersham PharamciaBiotech) Cy5 nonfunctional dye solid; and the third cartridge was loadedwith 1.5 mL of 0.2M sodium carbonate buffer, pH 9.97.

Cy5 is a fluorescent dye that contains a functional group that cancovalently attach the dye to a protein such as BSA.

The first and second cartridges were inserted into one of the dualbarrel syringe housings. The third cartridge and a fourth cartridge wereinserted into the other syringe housing. The third and fourth cartridgeswere empty when inserted into the housing.

The plungers of second and fourth cartridges were immobilized. Theplunger of first cartridge was not immobilized. Positive pressure wasapplied to the plunger of the third cartridge, thereby forcing sodiumcarbonate buffer that was loaded in the plastic tubing into the firstcartridge, which contained the BSA solid. The BSA dissolved in thebuffer solution entering the first cartridge, thereby forming a BSAsolution.

The plungers of the third and fourth cartridges were immobilized and theplunger of second cartridge was released from immobilization. Positivepressure was applied to the plunger of the first cartridge, therebytransferring the BSA solution to the second cartridge. The FLORORLINKCy5 dye was immediately solubilized by the BSA solution and the solutionturned deep blue. The flow was reversed to further mix the reactionsolution. The solution was allowed to react for 20 minutes, therebygenerating dye-labeled BSA.

Dye-labeled BSA was purified by separation using Sephadex G-25 (PD-10column available from Amersham Pharmacia Biotech AB, Uppsala, Sweden). Awell-defined separation between the dye-labeled BSA and the unreacteddye was observed on the column while eluting with water.

Example 6 Immobilized Chemical Reaction

This example demonstrates the enhancement of a chemical reaction on animmobilized matrix when the analyte is passed through an immobilizedmatrix multiple times. In this example, a protein solution was passedthrough a matrix containing immobilized trypsin. The trypsin cleaves theprotein in the solution, thereby generating peptide fragments.

An immobilized chemical reaction system was constructed and used foreach of two runs, a single-pass run and a multi-pass run. The system wassimilar to that described in Example 1, with the following exceptions:the glass column was packed with trypsin (TPCK treated) immobilized onbeaded agarose (available from Sigma, St. Louis, Mo.) and wasequilibrated with 100 mM Tris, pH 8.2; 1 mL of a BSA solution was loadedinto a first glass cartridge; and 2.5 mL 100 mM Tris, pH 8.2 was loadedinto a second glass cartridge.

The BSA solution was prepared by dissolving bovine serum albumin (BSAavailable from Sigma Chemical Co., St. Louis, Mo.) in 100 mM Tris, pH8.2 to an approximate concentration of 5 mg/mL.

The first and second cartridges were inserted into one dual barrelsyringe housing. A third cartridge and fourth cartridge were insertedinto another dual barrel syringe housing attached to the other end ofthe glass column. The third and fourth cartridges were empty wheninserted into the housing.

In the single-pass run, the BSA solution was introduced into thebead-packed column from the first cartridge by manipulation of theplunger rods as previously described. However, the BSA protein solutionwas allowed to react with the immobilized trypsin beads for 65 minutesat 23° C. After 65 minutes the column containing the BSA protein/peptidesolution was washed with the buffer solution from the second cartridge.The wash was performed as previously described. Thus, all of thetrypsinized BSA solution was collected in the third cartridge.

In the multi-pass run, the BSA solution was passed back and forththrough the bead-packed column 31 times at a rate of approximately 0.5mL/min. The column was maintained at a temperature of 23° C. during the31 passes. After the 31 passes, all of the trypsinized BSA solution wascollected in the third cartridge.

Approximately 3.5 mL of trypsinized BSA solution was collected in eachrun. Analysis of the extent of proteolysis was accomplished using sizeexclusion chromatography (SEC). Due to the lack of a chromophore in thesample, fractionation of protein and unreacted dye fractions using SECwere accomplished by analogy to the following standard.

In order to approximate the BSA solutions collected in the single-passrun and the multi-pass run, above, but have a chromophore present, asolution of dye-labeled BSA was prepared. It was assumed that all of theBSA from the original sample solution (5 mg) was collected in thecollected solutions (3.5 mL) of each run. Thus, 5 mg of dye-labeled BSA(albumin, bovine-sulforhodamine 101 acid chloride, Sigma, St. Louis,Mo.) was dissolved in 3.5 mL 100 mM Tris, pH 8.2. The resultingdye-labeled BSA standard solution approximated the SEC characteristicsof the solutions collected in the single-pass run and the multi-passrun.

The rhodamine-labeled BSA solution was applied to a Sephadex G-25 column(PD-10 column; Amersham Pharmacia Biotech AB, Uppsala, Sweden). Awell-defined separation between the rhodamine-labeled BSA and unreacteddye was observed on the column while eluting with 100 mM Tris, pH 8.2.The leading band contained rhodamine-labeled BSA and was collected in avolume of 7 mL. Elution of the unreacted dye required a collectionvolume of 20 mL.

Based on this standard, the solutions collected in the single-pass runand the multi-pass run were individually separated using SEC. For eachseparation, a 7 mL fraction (the non-proteolyzed protein fraction) and a20 mL fraction (the proteolyzed peptide fraction) were collected.

The four fractions (the single-pass protein fraction and peptidefraction, and the multi-pass protein fraction and peptide fraction) weresubjected to one-dimensional gel electrophoresis and stained withCoomasie blue. A stained band appeared at approximately 68 kDa,corresponding to non-proteolyzed BSA, for the protein fractions and wasabsent for the peptide fractions, confirming that any non-proteolyzedBSA was limited to the protein fraction.

Matrix Assisted Laser Desorption Ionization—Time of Flight MassSpectrometry (MALDI-TOFMS) was conducted (Voyager, Applied Biosystems,Foster City, Calif.) on each of the solutions to determine the extent oftrypsin proteolysis of the four samples. Samples were analyzed on a goldmatrix by spotting 2 μL of each fraction, followed by 1 μL of matrixsolution (alpha-cyano 4-hydroxycinnamic acid in acetonitrile:water, 1:1with 1% trifluoroacetic acid) and analyzing the sample according to themanufacturer's instructions.

The analysis showed that more complete proteolysis of the BSA occurredin the multi-pass run than in the single-pass run. The BSA molecular ionpeak (˜67 kDa) obtained from the single-pass experiment wasapproximately twice the intensity as the molecular ion peak obtainedfrom the multi-pass experiment, indicating that more BSA was proteolyzedin the latter experiment.

Example 7 Affinity Immobilization of Two Components from One Sample

This example demonstrates the simultaneous removal of two humanproteins, albumin and IgG, from human serum using a single columncontaining a mixture of immobilized affinity reagents. The system wasconstructed as described in Example 2, with the following exceptions:the glass column was packed with a mixture of 600 μL Mimetic Blue SAP6XL beads and 400 μL Mabsorbent A2P beads (both available from ProMeticBioSciences Ltd., Isle of Man, British Isles) and was equilibrated with50 mM sodium phosphate buffer, pH 6.0 containing 0.9% NaCl; 1.0 mL of ahuman serum sample was loaded into a first glass cartridge; 2.5 mL and1.5 mL of a 50 mM sodium phosphate buffer, pH 6.0 were loaded into asecond and third cartridge respectively.

The human serum sample was prepared by centrifuging 1.0 mL of humanserum (Impath Predictive Oncology, Franklin, Mass.) in a microfuge tubefor 10 minutes at 6,000 rpm in a tabletop centrifuge (VWR Scientific,West Chester, Pa.). 200 μL of the supernatant was diluted with 1.8 mL of50 mM sodium phosphate buffer, pH 6.0 containing 0.9% NaCl in order toform the human serum sample that was loaded into the first cartridge.

The first and second cartridges were inserted into one dual barrelsyringe housing. The third and a fourth cartridge were inserted intoanother dual barrel syringe housing attached to the other end of theglass column. The fourth cartridge was empty when inserted into thehousing.

Positive pressure was applied to the plunger rod of the first cartridge,thereby introducing the starting human serum into the glass column andcontacting the serum with the mixture of beads containing mixedimmobilized affinity agents for albumin and IgG. The plunger rods of thesecond and third cartridges were immobilized, but the plunger rod in thefourth cartridge was not immobilized. Thus, liquid collected in thefourth cartridge as the plunger of the first cartridge was advanced.When the plunger rod of the first cartridge was fully advanced, all ofthe starting human serum had been introduced into the column.

Next, the plunger rods of the first and third cartridges wereimmobilized and the plunger rod of the second cartridge was releasedfrom immobilization. Positive pressure was applied to the plunger rod ofthe second cartridge so that the buffer solution loaded into the secondcartridge was introduced into the column, thereby washing the treatedserum from the column. Because the plunger rod in the fourth cartridgewas not immobilized, treated serum washed from the column was directedtowards and collected in the fourth cartridge.

Next, the plunger rods of the second and third cartridge wereimmobilized and the plunger rod of the first cartridge was released fromimmobilization. Positive pressure was alternately applied to the plungerrods of the fourth cartridge and first cartridge so that the treatedserum was passed back and forth through the bead-packed column eightmore times. Whenever positive pressure was being applied to the plungerrod of one of the first or fourth cartridge, the plunger rod of theother cartridge (“the passive cartridge”) was allowed to be displacedoutward with respect to the cartridge, thereby allowing the treatedserum to temporarily collect in the passive cartridge.

After a total of nine passes through the column most of the treatedserum had been collected in the fourth cartridge. The plunger rods ofthe first and fourth cartridges were immobilized and the plunger rods ofthe second and third cartridges were released from immobilization.Positive pressure was applied to the plunger rod of the third cartridgeso that the buffer solution loaded in the third cartridge was thenintroduced into the column, thereby washing the remaining treated serumfrom the column. The remaining treated serum and wash buffer werecollected in the second cartridge.

Thus, all of the treated serum was collected in the fourth cartridge andthe second cartridge. The contents of the second cartridge and thefourth cartridge were combined and the resultant volume of the combinedtreated serum was 4.5 mL.

A BCA protein assay (Pierce, Rockford, Ill.) was performed on both thestarting human serum sample and the treated serum, the combined contentsof the third and fourth cartridges. The starting human serum samplecontained 7.51 mg of total protein. The treated serum contained 1.26 mgof total protein. Therefore 6.25 mg of protein was retained in thecolumn.

The protein retained in the column was eluted by liquid chromatography(UPC-900, Amersham Pharmacia Biotech, Uppsala, Sweden) using 0.1 M CAPS(3-cyclohexylamino-1-propane sulfonic acid) buffer, pH 11.5. Theresulting eluent contained 6.23 mg of total protein according to a BCAprotein assay.

Samples of the starting human serum, treated serum and the column eluentwere analyzed by one dimensional gel electrophoresis using a 4-15%Tris-HCl Ready Gel (available from BioRad, Hercules, Calif.) in arunning buffer of Tris buffer containing glycine and sodium dodecylsulfate. The gel was also loaded with an HSA standard (Sigma ChemicalCo., St. Louis, Mo.) and protein standards (Broadband Standardsavailable from BioRad, Hercules, Calif.). Each sample was denatured bymixing 20 μL of sample with 40 μL cracking buffer (50 μL2-mercaptoethanol and 950 μL Laemmli sample buffer (BioRad). 40 μL ofeach denatured sample was loaded into the well of a separate lane of theReady Gel. The gel was run at 120 V for approximately 1 hour.

The resulting gel was stained for 1 hour using BioSafe Coumassie(BioRad) and destained in deionized water overnight. The most-intenselystained proteins in the starting human serum sample and the columneluent were consistent with the molecular weights for HSA, IgGheavy-chain, and IgG light-chain. The lane containing the column eluentalso contained additional proteins. However, the intensity of the stainfor the additional proteins was much less than the intensity of stainingof the HSA, IgG heavy-chain and IgG light-chain. No IgG heavy-chain orIgG light-chain was detected in the lane containing treated serum; arelatively light-intensity band of HSA was detected.

The complete disclosures of the patents, patent documents andpublications cited herein are incorporated by reference in theirentirety as if each were individually incorporated. In case of conflict,the present specification, including definitions, shall control.

Various modifications and alterations to this invention will becomeapparent to those skilled in the art without departing from the scopeand spirit of this invention. It should be understood that thisinvention is not intended to be unduly limited by the illustrativeembodiments and examples set forth herein and that such examples andembodiments are presented by way of example only with the scope of theinvention intended to be limited only by the claims set forth herein asfollows.

What is claimed is:
 1. A device for treating a liquid sample comprising:a firm chamber having a variable volume; a second chamber having avariable volume; a third chamber having a variable volume; aninterconnect comprising one or more passageways, each passageway engagedwith one or more chambers so that the interconnect provides fluidcommunication between the first chamber, second chamber and thirdchamber; and a treating substance that comprises a functionalized solidsupport or an affinity reagent included in at least one of the firstchamber, the second chamber, the third chamber, or at least onepassageway of the interconnect; wherein the device is configured so thatmovement of the liquid sample through the device is controlled bychanging the variable volumes of two or more chambers.
 2. The device ofclaim 1 further comprising a second treating substance provided in thefirst chamber, the second chamber, the third chamber or at least aportion of the interconnect.
 3. The device of claim 2 wherein the secondtreating substance comprises a filter, a membrane, a size exclusionmedium, a derivalized solid support, a reactant, an affinity reagent, ora buffer.
 4. The device or claim 1 wherein the interconnect comprisestwo more passageways.
 5. The device of claim 4 wherein the interconnectfurther comprises two or more treating substances so that a firsttreating substance is provided in a first passageway that is differentthan a second treating substance provided in a second passageway.
 6. Thedevice of claim 1 further comprising an actuator connected to at leasttwo chambers and is configured to regulate the variable volumes of theat least two chambers.
 7. The device of claim 1 wherein at least onechamber is reversibly removable from the device.
 8. The device of claim1 further comprising at least one detection element for detecting atleast a portion of the liquid sample.
 9. The device of claim 1 whereinthe detection element comprises a pH detector, a spectrophotometricdetector, a fluorescence detector, a conductivity detector or arefractive index detector.
 10. The device of claim 1 further comprisingat least one temperature-regulating element for regulating thetemperature of at least a portion of the interconnect or at least aportion of at least one chamber.
 11. The device of claim 1 furthercomprising at least one controllable element connected to at least onecontroller element.
 12. The device of claim 11 wherein the controllableelement comprises an actuator for regulating the variable volume of atleast one chamber, a detection element, or a temperature-regulatingelement.
 13. The device of claim 11 wherein the controller elementcomprises a programmable microprocessor.
 14. The device of claim 1further comprising at least one additional chamber having a variablevolume that is in fluid communication with at least a portion of theinterconnect.
 15. A method of treating a liquid sample comprising: a)providing an device that comprises: i) a first chamber having a variablevolume, ii) a second chamber having a variable volume, iii) a thirdchamber having a variable volume, iv) an interconnect comprising one ormore passageways, each passageway engaged with one or more chambers sothat the interconnect provides fluid communication between the firstchamber, the second chamber and the third chamber, and v) a treatingsubstance that comprises a functionalized solid support or an affinityreagent included in at least one of the first chamber, the secondchamber, tho third chamber or a portion of the interconnect, wherein thedevice is configured so that movement of the liquid sample through thedevice is controlled by changing the variable volume of two or morechambers; b) providing a liquid sample in the first chamber; and c)decreasing the volume of the first chamber and increasing the volume ofat least one other chamber, thereby contacting at least at portion ofthe sample with at least a portion of the treating substance so that thesample is at least partially treated by the treating substance, therebyalso directing at least a portion of the sample toward the at least oneother chamber.
 16. The method of claim 15 wherein the step of decreasingthe volume of the first chamber further comprises decreasing the volumeof the second chamber.
 17. The method of claim 15 wherein a treatedportion of the sample is directed into a collection chamber, and themethod further comprises the step of removing the collection chamberfrom the device.
 18. The method of claim 15 wherein increasing thevolume of at least one other chamber comprises increasing the volume ofthe second chamber, thereby directing at least a portion of the sampletoward the second chamber.
 19. The method of claim 18 wherein at least aportion of the sample directed toward the second chamber comprisessample that has been treated by the treating substance.
 20. The methodof claim 18 wherein the device further comprises a second treatingsubstance included in at least one of the second chamber, the thirdchamber, or a portion of the interconnect; and the method furthercomprises the step of decreasing the volume of the second chamber andincreasing the volume of the third chamber, thereby directing at least aportion of the at least partially treated sample toward the thirdchamber and causing the at least partially treated sample to be furtherat least partially treated by the second treating substance.
 21. Themethod of claim 15 wherein the device further comprises at least onecontrollable element connected to at least one controller elementwherein the controller element comprises a programmable processor, andat least one portion of the method is automated and controlled by theprogrammable microprocessor.